Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms

ABSTRACT

A fermentation process for making succinic acid in high concentrations employs a bacteria obtained from the rumen contents of cattle. A preferred bacteria is Bacterium 130Z (ATCC No. 55618).

FIELD OF THE INVENTION

This invention relates to a process for making succinic acid,microorganisms for use in the process and methods for obtaining thesemicroorganisms.

BACKGROUND OF THE INVENTION

Succinic acid and its derivatives are widely used as specialty chemicalswith applications in foods, pharmaceuticals, and cosmetics.

Commercial fermentations for other organic acids, such as citric andlactic acids, typically produce concentrations of 80 to 120 g/l.However, such fermentations for succinic acid usually produce much lowerconcentrations of less than 40 g/l.

The Glassner et al. U.S. Pat. No. 5,143,834 and the Datta et al. U.S.Pat. No. 5,168,055, disclose integrated processes for the production ofsuccinic acid employing the anaerobic bacterium, Anaerobiospirillumsucciniciproducens, which produces comparatively low concentrations ofsuccinic acid of approximately 35 g/l.

Although succinic acid is a common intermediate in the metabolic pathwayof several anaerobic microorganisms, such as Propionibacterium, nomicroorganisms are described in the literature which produce succinatein high concentrations. Well known species of rumen bacteria convertplant carbohydrates to fatty acids and to a very large extent succinicacid.sup.(1). Prevotella ruminicola and Ruminobacter amylophilus areexamples of well known species of rumen bacteria that produce majoramounts of succinic acid but in generally low yields. Unfortunately, thetypical rumen organisms cannot tolerate the presence of highconcentrations of succinic acid or its salts and tend to lyse aftercomparatively short fermentation times which makes them unsuitable forindustrial application. It appears that the accumulation of very highconcentrations of acids, such as succinic acid, or their salts is not anormal phenomenon for microorganisms and it is damaging to them.

There is a need for a fermentation process for succinic acid whichproduces higher concentrations in the fermentation broth and whichpermit an economical recovery of succinic acid. It also would beadvantageous to have microorganisms that can tolerate higher levels ofsuccinic acid and its salts. It would be advantageous to have a methodof obtaining this type of organism.

SUMMARY OF THE INVENTION

The objects of the invention are to disclose a novel process for makingsuccinic acid in high concentrations, microorganisms that are useful inthat process and a method for obtaining such microorganisms.

The method of the present invention for the production of succinic acidbasically comprises, providing an aqueous fermentation medium containinga submerged culture of a microorganism and a source of assimilablecarbon, such as a carbohydrate; and cultivating said organism underanaerobic conditions or aerobic conditions in the presence of carbondioxide to form succinic acid in a concentration of at least about 50g/l in said fermentation medium.

The novel microorganisms of the present invention are ionophoreresistant species of rumen bacteria obtained from cattle. These speciesare more resistant to inhibition by succinic acid and produce a higherconcentration of succinate in a fermentation process than can beobtained in a corresponding fermentation using other succinate producingspecies under otherwise identical conditions.

The preferred method of obtaining strains for use in the method of thepresent invention for producing succinic acid comprises growing bacteriafrom the rumen contents of cattle in an aqueous fermentation medium withdisodium fumarate (about 5 g/l to about 30 g/l) and an antibioticionophore (about 8 mg/l to about 32 mg/l), which favors the growth ofthe succinate producing organisms; and, then isolating from the culturemedium the colony of the strain which produces succinic acid in thehighest concentration.

A preferred microorganism for use in the method is an isolated,biologically pure culture consisting essentially of Bacterium 130Z (ATCCNo. 55618).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

In the preferred practice of the present invention, a substantially pureculture of the Bacterium 130Z (ATCC 55618) is grown at a controlled pHbetween about 6.0 to about 7.2 in a fermentor on a medium containing asource of fermentable carbon containing a carbohydrate and othernutrients, such as corn steep liquor, under a partial pressure of atleast about 0.1 atmosphere CO₂ until the fermentation broth contains atleast about 50 g/l of succinic acid.

The source of fermentable carbon used in the practice of this inventioncan be any carbohydrate that is fermented by the strain of bacteriaused. These carbohydrate sources include dextrose, sucrose, fructose,lactose, soluble starches, and corn syrups. The fermentation isconducted in an aqueous medium in the presence of dissolved carbondioxide. Other nutrients and growth factors needed for the growth andthe reproduction of the microorganism employed also are added to themedium.

The concentration of carbohydrate in the medium is between about 20 g/lto about 150 g/l, preferably between about 90 g/l and about 120 g/l.Carbohydrate concentrations above about 70 g/l give solutions with suchhigh osmotic pressures that the organisms usually used to producesuccinic acid do not grow well; however, the strains of the presentinvention will tolerate such conditions.

Carbon dioxide can be supplied to the fermentation medium in variousways. The medium can be sparged with CO₂ gas. The fermentation can berun in a pressurized reactor which contains carbon dioxide atsuperatmospheric pressure. The CO₂ can be mixed with other gases as longas the gases employed do not interfere with the growth and metabolism ofthe organism employed. Carbon dioxide can also be supplied to thefermentation medium by the addition of carbonates or bicarbonates whichgenerate this gas under the conditions of the fermentation. The mediumshould contain dissolved CO₂ in equilibrium with a minimum of about 0.1atmosphere partial pressure of carbon dioxide. In the preferredembodiment, the medium is saturated with carbon dioxide and theatmosphere contains about 0.3 atmosphere partial pressure of carbondioxide or higher.

In order to obtain good production of succinate salt, the pH-of themedium is maintained in the range of from about 7.2 to about 6.0. The pHis conveniently maintained by the addition of alkaline carbonates,alkaline earth hydroxides, or mixtures thereof.

The fermentation process of this invention is carried out at atemperature between about 25° C. and about 40° C. For example, theoptimum growth of Bacterium 130Z is at about 38° C. Since it is afacultative anaerobe, fermentations using Bacterium 130Z can be carriedout under either anaerobic conditions or in the presence of air andcarbon dioxide in a medium which has been sterilized by heat or othermeans well known in the fermentation art.

The preferred Bacterium 130Z (ATCC No. 55618) has been demonstrated tobe capable of producing high concentrations of succinic acid (about 50to about 80 g/l) with a high productivity.

The Bacterium 130Z is obtained as previously described and as morespecifically described in the description of the experimental work.

MATERIALS AND METHODS

organism

The succinic acid producing strain Bacterium 130Z (ATCC No. 55618) wasisolated in a Michigan Biotechnology Institute (MBI) laboratory frombacteria from the rumen contents of fistulated cattle. Other succinicacid producing organisms used for comparison purposes wereAnaerobiospirillum succiniciproducens ATCC 53488, Ruminobacteramylophilus DSM 1361, Succinivibrio dextrinosolvens ATCC 19716,Prevotella ruminocola ATCC 19188 (American Type Culture Collection(ATCC), Rockville, Md.; German collection of Microorganisms and CellCultures (DSM), Braunschweig, Germany). chemicals

Chemicals were obtained from Sigma Chemicals, St Louis Mo. or AldrichChemical, Milwaukee, Wis. unless otherwise stated. Gases and gasmixtures were supplied by Linde (Michigan Welding, East Lansing Mich.).For anaerobic conditions, oxygen was scrubbed out by passage over heatedcopper filings. enrichment and isolation

Source: Rumen contents were collected from fistulated cattle on variousfeeding regimens. Samples were transported to MBI and transferred tovials for storage under nitrogen at 4° C.

Enrichment and isolation media: The standard salts (SS) were NaCl 1.0,K₂ HPO₄ 3.0, MgCl₂.6H₂ O 0.2, CaCl₂ H₂ O 0.2, (NH₄)₂ SO₄ 1.0 g/l. Forenrichments, 10 ml of SS was used with corn steep liquor (CSL) 20.0 g/l(Corn Products Corporation, Englewood Cliffs, N.J.). The same medium wasused for isolation of pure cultures but with the following additions:B₁₂ 1 mg; biotin, folic acid 20 mg; thiamine, riboflavin, niacin,pantothenate, p-aminobenzoate, and lipoic acid 50 mg; B₆ 100 mg; sodiumacetate 1.36 g; xylose 1 g; methyl butyrate, valerate, isobutyrate,isovalaerate 0.2 ml; 1,4-napthoquinone 10 mg; and hemin 500 mg/l. Forplating Bacto yeast extract 5.0 g and Bacto peptone 10.0 g/l weresubstituted for the CSL and 100 ml was dispensed into vials containing1.8 g of Bacto agar and 1.0 g of MgCO₃. All media were gassed anddispensed under CO₂ /N₂ (5%/95%). Plates were poured in an anaerobicglove bag (Coy Inc, Ann Arbor, Mich.). Glucose or maltrin (20 g/l) wasadded as substrates to the enrichment and isolation media. Disodiumfumarate (15 g/l) was added to the enrichment medium and plates forisolation. Stock solutions of Lasalocid (Sigma Chemical, St. Louis,Mo.), an antibiotic ionophore, were prepared in ethanol (10 mg/ml) andadded to enrichment and isolation plates to give a concentration of 16μg/ml.

Isolation and screening: An enrichment train was established with a 10%inoculum, and subcultures were made after 18 hours of incubation at 37°C. Samples from the third enrichment vials were diluted (10⁻⁶) inphosphate buffered saline and 0.1 ml aliquots were spread on plates inthe glove bag. Plates were incubated anaerobically in an Oxoid AnaerobicJar (Oxoid Ltd., Basingstoke, England) with a 100% CO₂ atmosphere. After24 to 48 hours single well isolated colonies were picked with a 22 gaugeneedle and washed into vials of glucose isolation medium. Primaryisolates were grown 24-36 hours in CSL medium. Samples from each vialwere analyzed by HPLC and screened for succinic acid production.

analysis

The organic acid fermentation products were determined usinghigh-performance liquid chromatography (HPLC).sup.(2). A waters Model600 HPLC system with a BioRad HPX-87H column and a Waters Model 410Refractive Index detector were used in this analysis. Carbohydrate alsowas determined by the same HPLC methods. Peptone yeast glucose (PYG)fermentation product determination was by gas liquidchromatography.sup.(3).

biochemical

Enzymatic activities were determined with the Rapid Ana II System(Innovative Diagnostics, Atlanta, Ga.), with cells grown on Brain HeartInfusion (BBL, Cockeysville, Md.). The API CH50 (Analytab Products,Plainview, N.Y.) was done as directed using cells grown on Rabbit BloodAgar plates (BBL, Cockeysville, Md.), aerobically with extra CO₂.Cellular Fatty Acids were determined using the Microbial IdentificationSystem (MIDI, Newark, Del.) and software for anaerobic and aerobicbacteria. Cells were grown anaerobically in PYG and aerobically usingTripticase Soy Agar (BBL). Peptone yeast (PY) medium was made asdescribed in the VPI manual.sup.(3).

The test for fumarate reduction was done in a manner ofHollander.sup.(4). Pressure tubes (Belco Glass, Vineland, N.J.) with 10ml of PBB medium was used. PBB medium contained NaCl 0.9, MgCl ₂.6H₂ O0.2, CaCl₂.2H₂ O 0.1, NH₄ Cl 1.0 g/l and resazurin 3, CoCl₂.6H₂ O 1.7,FeSO₄.7H₂ O, MnCl₂.4H₂ O, CaCl₂.2H₂ O, ZnCl₂ 1.0, Na₂ SeO₃ 0.17,CuCl₂.2H₂ O 0.2, NiSO₄.6H₂ O 0.2 6, Na₂ MoO₄, H₃ BO₃ 0.1 mg/l. Bactoyeast extract (1.2%) was added along with 0.2 ml phosphate buffer. Thephosphate buffer was KH₂ PO₄ 150 and K₂ HPO₄ 290 g/l. A 0.1% inoculumwas used and incubation was stationary at 37° C.

serum vial cultures

Serum vial culture technique was used and transfers were made bysyringe(5). MgCO₃, 20-80 g/l, was added to the vials for pH maintenance.Carbon dioxide dependeny tests were done in 158 ml serum vialscontaining 50 ml of FB medium(6) and 1 ml phosphate buffer. All vialswere incubated at 37° C. in a model G25 Incubator Shaker (New BrunswickScientific, Edison, N.J.). fermentation

One liter batch fermentations were conducted in 2-liter MultiGenfermenters (New Brunswick Scientific, Edison, N.J.). The temperature wascontroled at 39° C. Carbon dioxide was sparged at 0.05-0.1volume/volume/minute. The culture was stirred at 300 rpm with flat bladeturbine impellers. The pH was automatically controlled (Chemcadet, ColePalmer, Chicago, Ill.) with the addition of 10N NaOH, or 5.5M Na₂ CO₃,or combinations of NaOH and Na₂ CO₃. The medium was SS plus CSL and/oryeast extract, with other additions and modifications as stated.

Results

Enrichment and Isolation

Ionophore/fumarate enrichment vials produced high succinateconcentrations and isolates from these enrichments produced succinate atconcentrations greater than 30 g/l from glucose (Table 1).Concentrations over 30 g/l had not been seen with isolates from variousother enrichment schemes from various environments including the rumen.

Initial isolates were obligate anaerobes that produced succinic acidconcentrations higher than those produced by rumen species obtained fromculture collections. For example, isolate R413 produced 31.2 g/l ofsuccinic acid in a 1-liter fermenter and 37.9 in a 1-liter fedbatchreactor. They could initiate growth without added reducing agents andhad other desirable properties but were not ideal because yield was lostto the production of lactate. A second class of isolates werefacultative anaerobes typified by strain Bacterium 130Z. These strainsdid not produce lactate and produced succinic acid in a much higheryield than other rumen organisms and with the proper conditionsBacterium 130Z produced nearly 80 g/l succinic acid.

                  TABLE 1    ______________________________________    Acids production by isolates from ionophore/fumarate enrichment.    Isolate          Succinic   Lactate  Acetate                                     Formate                                            Pyruvate    ______________________________________    R413  .sup. 31.2 (65.5).sup.1                     14.3     6.6    0.7    0.0    130Z  44.4 (83.7)                     0.0      4.1    1.4    6.4    ______________________________________     .sup.1 succinic acid yield (wt %)

Bacterium 130Z

product concentration

The most distinguishing feature of Bacterium 130Z is its ability toproduce very high succinic acid concentrations. Bacterium 130Z producedmore than 60 g/l of succinic acid in less than 36 hours and accumulated75-79 g/l with continued incubation. No other known strain producesconcentrations this high (Table 2). Concentrations of nearly 80 g/l ofsuccinic acid were obtained in serum vial cultures containing 15 g/leach of corn steep liquor and yeast extract, 100 g/l glucose, and 80 g/lof MgCO₃. The (NH₄)₂ SO₄ was omitted.

                  TABLE 2    ______________________________________    Succinic acid produced by different microbial cultures.                             Succinic    Organism      Source     Acid (g/l)                                       Mode    ______________________________________    Bacterium 130Z                  isolate.sup.1,                             79        serum vial                  rumen    Bacterium 130Z                  isolate, rumen                             68        batch                                       fermenter    R413          isolate, rumen                             37.9      fed batch                                       fermenter    A. succiniciproducens                  ATCC 53488,                             37.6      batch                  dog                  fermenter    Ruminobacter  DSM 1361,  11.9      batch    amylophilus   rumen                fermenter    Succinivibrio ATCC 19716,                             26.7      batch    dextrinosolvens                  rumen                fermenter    Prevotella ruminocola                  ATCC 19188,                             18.9      batch                  rumen                fermenter    ______________________________________     .sup.1 Ionophore/fumarate enrichment isolates.

osmotolerance

Bacterium 130Z is tolerant to high succinate concentrations (Table 3).It grows in medium saturated with magnesium succinate and it producessuccinic acid in this medium despite any kinetic or equilibriumdisadvantage this presents. Bacterium 130Z produced succinic acid in afermenter with an initial concentration of 49 g/l disodium succinate,producing a concentration over 100 g/l in 39 hours. Well known succinateproducing strains do not tolerate product concentrations this high. A.succiniciproducens will not initiate growth in media containing 20 g/ldisodium succinate.

                  TABLE 3    ______________________________________    Tolerance to high succinate concentrations.              Initial Concentrations of Succinic Acid Salts                49 g/l     96 g/l     130 g/l    Organism    disodium   disodium   magnesium    ______________________________________    130Z        growth and growth and growth and                fermentation                           fermentation                                      fermentation    A.          no growth  no growth  no growth    succiniciproducens    ______________________________________

relationship to carbon dioxide

Bacterium 130Z is a capnophile. It requires an atmosphere enriched incarbon dioxide for rapid fermentative growth and development. Bacterium130Z's response to carbon dioxide concentrations is related to itsability to make high concentrations of succinic acid. Carbon dioxide isa substrate in the succinate pathway and production of highconcentrations of succinic acid causes a demand much higher than normalbiosynthetic activity. This strain is dependent upon carbon dioxidesupplementation for high succinate productivity.

relationship to nitrogen

Bacterium 130Z can obtain its nitrogen from organic sources. The normalconcentration of ammonium sulfate in the CSL containing medium is 1 g/l.Growth of Bacterium 130Z and succinic acid production is increased bythe elimination of the ammonium sulfate. Serum vial studies indicate atrend of increased succinate with the reduction of this salt (Table 4).Succinic acid concentrations of nearly 80 g/l were achieved in vialswith the complete elimination of ammonium sulfate. Performance in1-liter fermenters was also improved through the elimination of ammoniumsulfate and the extent of the effect may vary from lot to lot of CSL.Excess ammonium ion limits succinic acid production to less than 30 g/l.

                  TABLE 4    ______________________________________    Effect of 1 g/l ammonium sulfate on succinic acid production                     Succinic Acid Produced    (NH.sub.4).sub.2 SO.sub.4 (% of normal)                     (% of normal)    ______________________________________    25               123    50               109    100              100    150              87    200              79    ______________________________________

relationship to other nutrients

Bacterium 130Z grows fermentatively with glucose, some simple salts, anda small amount of yeast extract. It produces succinate and acetate asmajor products. The rate and extent of growth is markedly influenced bythe addition of yeast extract and casein hydrolysate. An extreme exampleis a fermentation with 20 g/l yeast extract and 2 g/l of caseinhydrolysate. Bacterium 130Z produced 41 g/l of succinic acid in 15hours.

Corn steep liquor (CSL), a byproduct of the corn wet milling industry,is an important fermentation medium constituent. The succinic acid yield(wt %) is improved by using a CSL based medium with an optimal amountsof yeast extract to stimulate rapid growth and fermentation. Nutrientsources can be combined to provide a high fermentation rate, highconcentration, and high yield. Acids production by Bacterium 130Z istractable by nutrient manipulation and responsive to optimizationefforts. Yeast extract and casein hydolysate speed up the fermentationand increase succinate concentration. CSL has the effect of improvingsuccinate yields. Optimum combinations can be arrived at empirically andmaybe varied to obtain the best economic results. High concentration andhigh yield are the results of using 20 g/l CSL and 1 g/l of yeastextract, the same results were obtained 10 hours sooner with 10 g/l CSLand 6 g/l yeast extract (Table 5).

                  TABLE 5    ______________________________________    Effect of nutrient on productivity.    Time  Corn steep liquor                       Yeast extract                                  Succinic acid                                           Yield    (hrs) (%)          (%)        (g/l)    (wt %)    ______________________________________    39.3  2            0.1        46.2     83    29.5  1            0.6        45.8     83    ______________________________________

relationship to oxygen

Bacterium 130Z is a facultative anaerobe that has a fermentativemetabolism. It is not sensitive to oxygen exposure as is A.succiniciproducens and other obligate anaerobes (Table 6). Fermentationmedia for Bacterium 130Z can be prepared in the standard manner withoutany special effort to exclude oxygen. Bacterium 130Z is tolerant toprotracted exposures to air and will grow well in the presence of air ifit is supplied with an increased partial pressure of CO₂.

                  TABLE 6    ______________________________________    Growth.sup.1 and fermentation after 60 minutes exposure to air.    Organism               Before   After    ______________________________________    Bacterium 130Z         +        +    Anaerobiospirillum succiniciproducens                           +        -    ATCC 53488    Prevotelia ruminicola ATCC 19188                           +        -    Ruminobacter amylophilus DSM 1361                           +        -    Succinivibrio dextrinisolvens ATCC 19188                           +        -    ______________________________________     .sup.1 Growth in anaerobically prepared media.

relationship to substrate

Bacterium 130Z can grow and produce succinic acid in aqueous mediacontaining more than 150 g/l dextrose, thus demonstrating an exceptionaltolerance to high substrate concentrations and to unfavorable osmoticconditions. A. succiniciproducens does not grow in media containing over70 g/l. Media containing 20 to 120 g/l dextrose are suitable forsuccinic acid production (Table 7). This organism also produces succinicacid from sucrose, lactose and byproducts such as whey.

                  TABLE 7    ______________________________________    Growth and succinic acid production with different substrate    concentrations.                 Initial Dextrose (g/l)    Organism       50         85     100    ______________________________________    Bacterium 130Z 41         65      79    A. succiniciproducens                   38         ng.sup.1                                     ng.sup.1    ______________________________________     .sup.1 no growth

relationship to pH

succinic acid production

Succinic acid production by A. succiniciproducens is markedly affectedby slight shifts in pH (Table 8). In contrast, Bacterium 130Z istolerant to a wider range of pH and therefore does not require strict pHcontrol. A pH of 6.2 to 7.0 is generally satisfactory for high succinateproduction and yield (Table 8).

                  TABLE 8    ______________________________________    Effect of PH on product formation.               Grams of Succinic Acid Produced.sup.1    pH ± 0.1  130Z.sup.2                          A. succiniciproducens    ______________________________________    6.2          50.8    43.5    6.8          56.6    20.2    7.2          41.8    14.7    ______________________________________     .sup.1 Calculated amount based on 1000 ml of fermentation medium.     .sup.2 Various nutrient conditions were employed and no pH optimum is     implied.

magnesium neutralization

Succinic acid can be produced through fermentation using magnesiumcarbonate for neutralization and pH maintenance. Media containing 80 g/lof magnesium carbonate can beused to produce 80 g/l succinic acidconcentrations.

sodium neutralization

Succinic acid can be prepared through fermentation using sodiumhydroxide or sodium carbonate solutions for acid neutralization. The useof Na⁺ ion is desirable in a succinic acid process with recovery basedon water splitting electrodialysis. Solutions of NaCO₃ or NaOH wereemployed for pH control with many Bacterium 130Z fermentations and bothsodium salts were satisfactory for pH control. The sodium neutralizedfermentations of Bacterium 130Z and A. succiniciproducens are comparedin Table 9.

                  TABLE 9    ______________________________________    Comparison of sodium neutralized fermentations.            Time (hrs)                    Succinic acid (g/l)                                  Yield wt (%)    ______________________________________    Bacterium 130Z              29.5      45.8          83    A. succinici-              29.5      34.1          87    producens    ______________________________________     130Z: 1% CSL, 0.6% YE and 2 g/l MgCO.sub.3. Neutralization was with 10N     NaOH; A. succiniciproducens: 2% CSL, and tryptophan 25 ppm. Neutralizatio     was with 5.5M NaCO.sub.3.

pH tolerance

A 10N solution of NaOH was used for neutralization in many Bacterium130Z fermentations. In both the 0.3 and 1.0 liter fermenters, dosingwith 10N NaOH results in momentary pH heterogeneity, but Bacterium 130Zwas tolerant of pH control with 10N NaOH even in these small vessels.Bacterium 130Z was also tolerant of pH upsets. When alkali flow wasinterrupted overnight the pH desended to pH 5.1, Bacterium 130Z grew andconsumed an additional 44 grams of dextrose after control wasreestablished.

Description of Bacterium 130Z

Bacterium 130Z

Bacterium 130Z is a novel succinate producing strain with propertiessuperior to any currently described, and it supplants A.succiniciproducens in an evolving succinic acid fermentation process.The most salient feature of this organism is that it produces largeamounts of succinate from glucose and other carbohydrates. Over 93 g/lof magnesium succinate was produced from glucose in serum vials and 73g/l of disodium succinate was produced in a 1-liter fermentor. Bacterium130Z is remarkably tolerant to high succinic acid salt concentrations.It will grow and produce succinate in a medium containing 96 g/ldisodium succinate and it will grow in a medium saturated with 130 g/lmagnesium succinate. Bacterium 130Z produces succinate over a widerrange of pH than A. succiniciproducens. The rate and efficiency withwhich carbohydrate is converted to succinate is flexible and can becontrolled by changes in the medium. Succinic acid yields of 83-87 (wt%) have been attained.

Morphology

In CSL containing media Bacterium 130Z produces bacillary orcoccobacillary forms. Bacterium 130Z is Gram negative. It producesnonmotile, nonsporeforming, pleomorphic rods. In rapidly growingcultures cells were smaller, had a regular rod shape, and were sometimesseen in short chains of four to six cells. Growth on glucose platingmedia was rapid and colonies were yellowish with a frosted glassappearance.

Growth and metabolic products

Bacterium 130Z is a chemoorganotoph. It grows on many commonheterotrophic media. It grew rapidly at 37°-39° C. Succinic and aceticacids are produced as the major metabolic products after 48 hours growthin PYG medium. Approximately 30 meq/l of succinic acid and 10 meq/l ofacetic acid are produced in this poorly buffered medium. Small amountsof pyruvic and formic acid are also produced along with a trace ofoxalacetic acid. NaCl (0.5%) stimulates the growth of Bacterium 130Z inPYG. Bacterium 130Z grows fermentatively in PY medium by utilizing 11different carbohydrates (Table 10). Cultures of Bacterium 130Z remainedviable for up to 1 month at 4° C.

                  TABLE 10    ______________________________________    Growth and non-growth substrates for Bacterium 130Z.           Carbohydrates    ______________________________________    growth   arabinose, fructose, galactose, glucose, lactose,             maltose, mannitol, mannose, sucrose, xylose,             salicin.    nongrowth             inositol, melibiose, rhamnose, raffinose, trehalose    ______________________________________

Fumarate reduction

The fumarate reductase test follows changes in turbidity in a mediumcontaining fumarate but lacking carbohydrate (Table 11). It is ameasurement of the terminal step in the succinate pathway and theresults indicate the presence of strong fumarate reductase system inBacterium 130Z.

                  TABLE 11    ______________________________________    Growth response with disodium fumarate.    Incubation time   Growth with.sup.1    hours             50 mM   none    ______________________________________    0                 0.03    0.02    8.5               0.62    0.02    15.25             0.95    0.02    25                0.64    0.03    ______________________________________     .sup.1 600 nM

Biochemical

Based on Rapid AnaII, arginine aminopeptidase was present. Leucine,glycine, proline, phenylalanine, and serine aminopeptidase were absent.Arabinosidase, disaccharidase, β-galactosidase, α-glucosidase,β-glucosidase, α-galactosidase, α-fucosidase, acetyl-β-glucosaminidase,phosphatase, pyroglutamic acid arylamidase, tryptophanase (indole), andurease were absent.

Based on the API CH50 biotype, Bacterium 130Z is biochemically veryreactive and produces acid from the degradation of 27 of 49 substrates(Table 12).

                                      TABLE 12    __________________________________________________________________________    API 49 carbohydrate profile, CH50.    __________________________________________________________________________    0 CONTROL     - 18 MANNITOL        + 36                                           STARCH       +    1 GLYCEROL    + 19 SORBITOL        + 37                                           GLYCOGEN     +    2 ERYTHRITOL  - 20 a                       METHYL MANNOSIDE                                       - 38                                           XYLITOL      -    3 D ARABINOSE - 21 a                       METHYL GLUCOSIDE                                       - 39                                           GENTIOBIOSE  +    4 L ARABINOSE + 22 N ACERYL GLUCOSAMINE                                       - 40                                           D TURANOSE   -    5 RIBOSE      + 23 AMYGDALIN       + 41                                           D LYXOSE     -    6 D XYLOSE    + 24 ARBUTIN         + 42                                           D TAGATOSE   -    7 L XYLOSE    - 25 ESCULIN         + 43                                           D FUCOSE     -    8 ADONITOL    + 26 SALICIN         + 44                                           L FUCOSE     -    9 b      METHYL XYLOSIDE                  - 27 CELLOBIOSE      + 45                                           D ARABITOL   +    10      GALACTOSE   + 28 MALTOSE         + 46                                           L ARABITOL   -    11      GLUCOSE     + 29 LACTOSE         + 47                                           GLUCONATE    +    12      FRUCTOSE    + 30 MELIBIOSE       - 48                                           2 KERO GLUCONATE                                                        -    13      MANNOSE     + 31 SUCROSE         + 49                                           5 KETO GLUCONATE                                                        +    14      SORBOSE     - 32 TREHALOSE       +    15      RHAMNOS     - 33 INULIN          -    16      DULCITOL    - 34 MELEZITOSE      -    17      INOSITOL    - 35 RAFFINOSE       +    __________________________________________________________________________

Cellular fatty acids and DNA base composition

Bacterium 130Z produced a very restricted pattern of cellular fattyacids (Table 13). Hydroxylated and nonhydroxylated long chain fatty acidmethyl esters are present in whole-cell methanolysates. The predominantfatty acids were hexadecenoic acid (C_(16:1)) and hexadecanoic(C_(16:0)). Large amounts of hydroxylated fatty acid are typical of Gramnegative bacteria and 3-hydroxytetradecanoic (3OH-C_(14:0)) was presentin Bacterium 130Z along with the straight chain tetradecanoic acid(C_(14:0)).

                  TABLE 13    ______________________________________    Cellular fatty acid profile of Bacterium 130Z                    % area    ______________________________________    16:1 CIS 9 FAME   32.90    16:0 FAME         32.75    14:0 FAME         18.80    14:0 3OH FAME     13.00    18:1 c11/t9/t6 FAME                      2.56    ______________________________________

The DNA determined by an HPLC method.sup.(7) was 44.7-45.5 mol. % G+C.

In the method of the present invention for obtaining a ionophoreresistant microorganism (such as Bacterium 130Z) the medium which isemployed favors the growth of succinate producing organisms whileinhibiting those producing other acids such as butyrate. How the mediumworks is not completely understood. However, the medium should containthe following:

1. Disodium fumarate, to give an energetic advantage to the rumenorganisms with a strong pathway to succinate. In the final step of thepathway, fumarate is reduced to succinate and energy is conserved asATP.

2. An antibiotic ionophore, such as Lasalocid, which is usedcommercially to increase feed efficiency in cattle on high energy dietsand which selects for organisms that increase propionic acid in therumen.

3. Dissolved carbon dioxide, a catabolic substrate in the succinatepathway.

4. MgCO₃, to allow the growth of acidogenic strains by maintaining pHand providing a continuous supply of CO₂ /HCO₃ ⁻.

5. Carbohydrate, to obtain strains able to utilize the desired feedstock for succinic acid production.

The preferred medium does not merely obtain the well known rumenorganisms, it allows the development and isolation of organisms thatproduce succinate concentrations higher than those previously describedfor any organism.

It will be apparent to those skilled in the art that a number of changesand modifications can be made without departing from the spirit andscope of the invention. Therefore, it is intended that the inventiononly be limited by the claims.

References

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We claim:
 1. A biologically pure culture of a microorganism having allthe identifying characteristics of Bacterium 130Z (ATTC No. 55618).